doi:10.1016/j.jmb.2006.08.080
J. Mol. Biol. (2006) 364, 152–169
Substrate Recognition, Protein Dynamics, and
Iron-Sulfur Cluster in Pseudomonas aeruginosa
Adenosine 5′-Phosphosulfate Reductase
Justin Chartron 1 †, Kate S. Carroll 2 ⁎†, Carrie Shiau 4 , Hong Gao 2,3
Julie A. Leary 3 , Carolyn R. Bertozzi 2,4,5 and C. David Stout 1 ⁎
1
Department of Molecular
Biology, The Scripps Research
Institute, La Jolla,
CA 92037, USA
2
Department of Chemistry,
University of California,
Berkeley, CA 94720, USA
3
Department of Chemistry and
Molecular Cell Biology, Genome
Center, University of California,
Davis, CA 95616, USA
4
Department of Molecular and
Cell Biology, University of
California, Berkeley,
CA 94720, USA
5
Howard Hughes Medical
Institute, University of
California, Berkeley,
CA 94720, USA
APS reductase catalyzes the first committed step of reductive sulfate
assimilation in pathogenic bacteria, including Mycobacterium tuberculosis,
and is a promising target for drug development. We report the 2.7 Å
resolution crystal structure of Pseudomonas aeruginosa APS reductase in the
thiosulfonate intermediate form of the catalytic cycle and with substrate
bound. The structure, high-resolution Fourier transform ion cyclotron
resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis,
establish that the two chemically discrete steps of the overall reaction take
place at distinct sites on the enzyme, mediated via conformational flexibility
of the C-terminal 18 residues. The results address the mechanism by which
sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P.
aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for
catalysis. The structure reveals an unusual mode of cluster coordination by
tandem cysteine residues and suggests how this arrangement might
facilitate conformational change and cluster interaction with the substrate.
Assimilatory 3′-phosphoadenosine 5′-phosphosulfate (PAPS) reductases
are evolutionarily related, homologous enzymes that catalyze the same
overall reaction, but do so in the absence of an [Fe-S] cluster. The APS
reductase structure reveals adaptive use of a phosphate-binding loop for
recognition of the APS O3′ hydroxyl group, or the PAPS 3′-phosphate
group.
© 2006 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: APS reductase; [Fe-S] cluster; crystal structure; PAPS reductase;
enzyme mechanism
Introduction
Metabolic assimilation of sulfate (SO42−) from the
environment requires its reduction to sulfite (SO32−).
† J.C. and K.S.C. contributed equally to this work.
Present address: K. S. Carroll, Department of Chemistry
and Life Sciences Institute, University of Michigan, Ann
Arbor, Michigan 48109, USA.
Abbreviations used: APS, adenosine 5′-phosphosulfate;
FT-ICR, Fourier transform ion cyclotron resonance; PAPS,
3′-phosphoadenosine 5′-phosphosulfate; Trx, thioredoxin;
NCS, non-crystallographic symmetry; SAD, single
anomalous dispersion; MAD, multiple anomalous
dispersion.
E-mail addresses of the corresponding authors:
katesc@umich.edu; dave@scripps.edu
In many species of bacteria and plants this pathway,
which culminates in the biosynthesis of cysteine and
methionine, proceeds via adenosine 5′-phosphosulfate (APS)1–3 (Supplementary Data Figure 1). This intermediate is produced by the action of ATP
sulfurylase, which condenses sulfate and adenosine
5′-triphosphate (ATP) to form APS.4,5 The activated
sulfonucleotide is reduced to sulfite and adenosine
5′-phosphate (AMP) by APS reductase (Figure 1).6,7
This reaction requires reducing equivalents provided by the protein cofactor thioredoxin (Trx).
Humans lack the enzymes required for sulfate
reduction. Thus, APS reductase may be an attractive
drug target if the enzyme is required for bacterial
survival or virulence in vivo.3 NO and superoxide
are produced in response to Mycobacterium tuberculosis infection,8–11 and it is likely that the bacterium
has a mechanism of protection against these reactive
oxidants. Products of the reductive sulfate assimila-
0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
153
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 1. Reaction catalyzed by sulfonucleotide reductases.
tion pathway, such as mycothiol (biosynthesized
from cysteine), are excellent candidates for this
function. Consistent with this hypothesis, APS
reductase was identified in a screen for essential
virulence genes in Mycobacterium bovis BCG.12
Moreover, it was demonstrated recently in a murine
model of tuberculosis infection that the APS
reductase gene (CysH) is essential for the bacteria
to survive during the persistence phase. 13 No
antibiotic is currently available to target this part
of the bacterial lifecycle, so inhibitors of APS
reductase could represent the first drugs that
address the latent phase. Toward this end, it is
essential to obtain high-resolution structural information for this enzyme in complex with its
sulfonucleotide substrate.
Interestingly, not all organisms that assimilate
sulfate reduce APS as the source of sulfite. Through
divergent evolution, some organisms, such as
Escherichia coli and Saccharomyces cerevisiae, reduce
the related metabolite 3′-phosphoadenosine 5′phosphosulfate (PAPS), which is produced by APS
kinase from ATP and APS (Figure 1; and Supplementary Data Figure 1).14–17 Biochemical, spectroscopic and mass spectrometry investigation of
sulfonucleotide reductases in both APS-dependent
and PAPS-dependent bacteria have recently identified a two-step mechanism, in which the sulfonucleotide undergoes nucleophilic attack to form an
enzyme-thiosulfonate (E-Cys-Sγ–SO3–) intermediate,
followed by release of sulfite in a thioredoxindependent manner (Figure 2).6,18 Hence, APS and
PAPS reductases perform the same chemistry and,
overall, their primary sequences are homologous
(Figure 3; and Supplementary Data Figure 2) despite
the difference in substrate specificity.6,18
Efficient reduction of the thiosulfonate bond
requires the protein reductant Trx. Thus, in the
absence of Trx, the sulfite remains covalently
attached to an essential cysteine residue near the C
terminus.6,19 Small-molecule reductants with redox
potentials comparable to Trx, such as dithiothreitol
(DTT) and β-mercaptoethanol, release the intermediate from the unfolded polypeptide at elevated
temperatures, but do not support multiple turnover
for the active, folded catalyst.6 These data suggest
that the second half of the catalytic cycle is
dependent upon protein–protein interaction between Trx and APS reductase, most likely to
facilitate conformational rearrangements. The molecular details of such changes are not known, but
are essential for understanding how sulfonucleotide
reductases preserve the thiosulfonate intermediate
until interaction with Trx and subsequent reduction.
A central feature that distinguishes APS from
PAPS reductases is the presence of a conserved
cysteine motif, CC-X∼80-CXXC, which occurs in
addition to the universally conserved catalytic
cysteine residue (Figure 3; and Supplementary
Data Figure 2).3,16 The presence of this motif is
Figure 2. Mechanism of sulfonucleotide reduction.
154
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 3. Primary sequences for APS reductases from P. aeruginosa, M. tuberculosis, and B. subtilis, and for PAPS
reductase from E. coli, are shown based on alignment of 38 APS and 34 PAPS reductases (Supplementary Data Figure 2).
Secondary structure elements are denoted for P. aeruginosa APS reductase, and residues within the P-loop (60-66), LDTG
motif (85-88), and Arg-loop (163-171), are boxed. The bar graph indicates the degree of conservation based on all 72
sequences (Supplementary Data Figure 2). Strictly conserved residues are outlined in black; six additional residues
conserved in 38 APS reductases that ligate or interact with the [4Fe-4S] cluster are boxed in gray.
correlated with the presence of a [4Fe-4S] cluster
and, when the cluster is present, it is required for
catalytic activity.2,6,18,20,21 PAPS-specific reductases
lack the cysteine motif and therefore lack the [Fe-S]
cluster.1,16 As a result, the evolutionary divergence
that has segregated specificity toward APS versus
PAPS, but maintained a common two-step chemical
transformation, has resulted also in APS-specific
enzymes that contain an [Fe-S] cluster. This raises
the question of whether the cluster is involved in the
chemical mechanism of APS reduction.2,6,18,20,31 In
this regard, it is interesting to note that the
occurrence of sequential cysteine residues in the
sequence motif associated with the [4Fe-4S] cluster
in APS reductase is highly unusual, and has led to
doubts as to whether both cysteine residues coordinate the cluster.2,20 Structural definition of cluster
coordination is therefore an important step toward
understanding its functional role.
Here, we address outstanding questions regarding substrate recognition, protein dynamics during
the catalytic cycle, and cluster coordination through
structure determination of Pseudomonas aeruginosa
APS reductase with APS bound, and through
biochemical, kinetic and Fourier transform ion
cyclotron resonance (FT-ICR) mass spectrometry
experiments with both P. aeruginosa and M. tuberculosis APS reductases. The features of the structure,
and the dynamic properties of the enzyme, indicate
that APS reductase must undergo significant conformational rearrangement in formation of the
thiosulfonate intermediate, and in subsequent reduction by thioredoxin, and that the two discrete
steps of the overall reaction occur at distinct sites on
the enzyme. Furthermore, the structure establishes
coordination of the [4Fe-4S] cluster by a tandem
cysteine motif, Cys139 and Cys140, together with
Cys228 and Cys231. We propose that the unusual
conformation associated with ligation by adjacent
cysteine residues might allow for flexible coordination at an iron atom and be associated with
conformational changes during the catalytic cycle.
Finally, the structure reveals a role for a phosphatebinding loop (P-loop) in substrate recognition and
provides a molecular rationale for substrate specificity by sulfonucleotide reductases.
Results
Protein fold, APS site, and [4Fe-4S] cluster
The crystal structure of P. aeruginosa APS reductase is the first sulfonucleotide reductase to be
determined with a substrate bound, and it is the
first containing a [Fe-S] cluster. The protein
monomer folds as a single domain with a central
six-stranded β sheet with five parallel strands, and
one strand (β5) anti-parallel (Figures 3, and 4(a) and
(b)). Strands of the central β sheet are interleaved
with seven α-helices that pack against both sides of
the β sheet. Residues 220–249 comprise a pair of
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
consecutive β-loops; this structural element caps
the space between α-helices flanking the β-sheet to
create a deep active site cleft. The substrate APS
extends across the C-terminal ends of the β strands
(Figure 4(a)). Opposite the nucleotide at one end of
the active site cleft is the [4Fe-4S] cluster with
standard tetrahedral geometry. The cluster is
ligated by four cysteine residues, Cys228 and
Cys231 positioned at the tip of a β-loop, and the
tandem pair Cys139 and Cys140 within a long,
kinked helix, α6A. Three additional elements define
the active site: the P-loop between β1 and α3
(residues 60-66), the LDTG motif following β2
(residues 85–88), and the Arg-loop between β4
and β5 (residues 162–173). The C-terminal segment
of residues 250–267, which carries the catalytically
essential Cys256, is disordered, but could be
positioned above the active site cleft.
Quaternary structure
P. aeruginosa APS reductase crystallized as tetramer, consistent with its oligomeric state in solution
(Figure 4(c)).6 In accord with the non-crystallographic symmetry (NCS) observed for the [Fe-S] clusters,
the tetramer has strict twofold symmetry but only
pseudo-222-fold symmetry. The strict twofold relates
the AB dimer onto the CD dimer (root-mean-square
deviation (rmsd) 0.14 Å for Cα atoms) (vertical axis in
Figure 4(c)), while the pseudo-twofold axes relate
individual subunits, i.e. A onto B, A onto D, etc., with
rmsd values of 0.77–0.79 Å. The two tetramers in the
asymmetric unit are very similar (rmsd for Cα atoms
of ABCD onto EFGH 0.15 Å). The asymmetry of the
AB and CD dimers is manifested in the relative
occupancy of APS in the B and D versus A and C
subunits (Figure 4(c)). The tetrameric arrangement
would allow interaction with Trx near the active site
of any given subunit.
The quaternary structure positions the nucleotides
in subunits B and D ∼35 Å apart. At the AB and CD
interfaces, two loops are juxtaposed at Gly222 and
Pro237, while at the AC and BD interfaces there are
extensive contacts between β3 strands and α5 helices
on opposing subunits (Figure 4(c)). The closest
contacts at each interface involve conserved glycine
residues: Gly222 (Cα–Cα distance 3.5 Å); and Gly88
in the LDTG motif (Cα–Cα distance 4.4 Å). Conservation of these glycine residues suggests that other
bacterial APS reductases may also be tetramers
(Supplementary Data Figure 2). In this respect, two
of three proline residues within β3 and α5 are not
conserved in the M. tuberculosis, Mycbacterium
smegmatis, and Rhizobium meliloti enzymes, which
function as monomers.6
State of the enzyme in crystals and mobility of
C-terminal residues
In the absence of Trx, APS reductase reacts with
APS, producing the thiosulfonated adduct of the
conserved cysteine residue Cys256 (Figure 2).6,18
Since formation of the covalent intermediate stabi-
155
lizes the cluster toward oxidation and loss of
activity,18 we anticipated that its formation would
constrain enzyme conformation and favor crystallization. However, the C-terminal residues beyond
Glu249 are disordered in all eight subunits in the
asymmetric unit, while APS is observed in four of
the active sites (subunits B, D, F, and H) (Supplementary Data Figure 3). Because the crystals were
grown with >60-fold excess of APS over enzyme, we
hypothesized that the C-terminal segment carrying
the thiosulfonate adduct is displaced from the active
site by unreacted APS.
To test this hypothesis, we carried out several
experiments. First, the stability of the thiosulfonate
intermediate was tested against small-molecule
reductants of various reduction potential. As observed previously, only Trx is able to catalyze the
second step of the reaction (Supplementary Data
Table 1). In particular, neither 10 mM DTT nor
10 mM dithionite, which have lower reduction
potentials, were able to release detectable sulfite
during the incubation time of the assay. Hence, the
presence of reductants in crystallization drops
(2.3 mM DTT and 2.5 mM dithionite) would not be
expected to cleave the thiosulfonate.
Second, washed and re-dissolved crystals were
assayed by mass spectrometry, showing quantitative sulfonation (31,359.8 Da calculated, 31360.1 Da
theoretical) consistent with the expectation that the
enzyme should form the enzyme-thiosulfonate
intermediate during crystallization (Supplementary
Data Figure 4(a)). Consideration of the anaerobic
conditions, concentrations of reagents present, and
rates, assure that the thiosulfonate can arise only
from enzymatic activity (Materials and Method). In
subunits B, D, F, and H, APS was sufficiently
ordered to visualize difference electron density
(Supplementary Data Figure 3), although the Bfactors were high (Table 1); in subunits A, C, E, and
G, extra density in the active site was observed, but
could not be modeled with confidence. Apparently,
sufficient APS was present to displace the sulfonated C-terminal residues in these subunits as well.
This interpretation was supported by the mass
spectrum of M. tuberculosis APS reductase, acquired
under non-denaturing conditions and after exposure to 20 μM APS, which showed the presence of
sulfonated enzyme with non-covalently bound
AMP, as well as sulfonated enzyme with noncovalently bound APS (Supplementary Data Figure
4(b) and (c)). Thus, the enzyme-thiosulfonate intermediate can still bind substrate. (Previously, at
concentrations of APS of 10 μM or less, sulfonated
enzyme with only AMP bound was observed.6)
Together, these data indicate that high concentrations of APS displace the Cys256 Sγ-SO3– intermediate
from the active site.
Third, to probe the conformation of the enzyme in
solution, M. tuberculosis APS reductase was subjected to limited trypsin proteolysis in the absence of
APS, and in the presence of equimolar APS; the
resultant peptides were purified by HPLC and
analyzed by mass spectrometry (Figure 5; and
156
Supplementary Data Figure 5). In the absence of
APS, essentially all M. tuberculosis APS reductase
was proteolyzed to lower molecular mass fragments
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
missing significant portions of the C terminus
(Figure 5; and Supplementary Data Figure 5(a)). In
the presence of stoichiometric APS, however, the
Figure 4. (a) The structure of
P. aeruginosa APS reductase comprises a central six-stranded β sheet
flanked by α-helices and loops that
create a deep active site cavity. The
substrate APS binds across the Cterminal ends of the β-strands, and
occupies the active site in half of the
independent copies of the protein in
the crystal (subunit B is shown). The
[4Fe-4S] cluster is ligated by four
cysteine residues at positions 139
and 140 on the long, kinked helix α6,
and at positions 228 and 231 at the
tip of a β-turn. (b) Secondary structure topology of P. aeruginosa APS
reductase indicating α-helix and βstrand elements, active site loops,
conserved residues Lys144 and
Cys256, the LDTG motif, and Cys
ligand connectivity to the [4Fe-4S]
cluster. (c) Triclinic crystals of P.
aeruginosa APS reductase contain
two tetramers in the asymmetric
unit. Each chain of the ABCD
tetramer is colored blue to red from
N terminus to C terminus (residues
27–249). Labeled residues are involved in inter-subunit contacts.
157
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 4 (legend on previous page)
major tryptic fragment comprised residues 55 to the
end of the protein including an intact, sulfonated Cterminal tail. A similar effect was found with P.
aeruginosa APS reductase (Figure 5; and Supplementary Data Figure 5(b)). In this case, proteolysis in the
absence of APS occurred primarily at Arg171 in the
Arg-loop, and at basic residues near the N and C
termini (Arg12, Lys254, Lys266) (Figures 3 and 5). In
the presence of APS, the major fragment consisted of
residues 13–266, which included the sulfonated
catalytic cysteine residue. In contrast to the protection afforded by equimolar substrate, addition of a
fivefold excess of APS resulted in significant cleavage at Arg171 and Lys254 (data not shown). Considering the results for M. tuberculosis and P. aeruginosa
APS reductase together, both the Arg-loop and the Cterminal tail are susceptible to proteolysis in the
absence of substrate; but in the presence of equimolar
substrate, thiosulfonate formation protects against
proteolysis by ordering these mobile elements.
However, in the presence of a large excess of substrate, as in crystals, or in solution, a third state exists
with APS bound and both the Arg-loop and the sulfonated C-terminal tail expelled from the active site.
Conformational states in steps of the reaction
We propose a model in which the C-terminal tail is
mobile: in the “open” form, APS can bind; in the
“closed” form with APS bound, the thiosulfonate
intermediate is formed. The intermediate is stable
with respect to small-molecule reductants, but is
reducible by Trx, which can interact with the
Cys256Sγ-SO3– to catalyze release of sulfite. At the
same time, formation of the intermediate protects the
C-terminal residues, as well as the Arg-loop, from
proteolysis. In the presence of excess substrate, as
during crystallization, APS binding displaces the
158
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Table 1. Crystallographic statistics
A. Crystals, unit cells, and data sets
Space group
Unit cell parameters
a (Å)
b (Å)
c (Å)
α (deg.)
β (deg.)
γ (deg.)
APSr SAD
APSr MAD
APSr native
P1
P1
P1
60.25
103.27
140.26
90.36
102.38
89.95
60.24
102.94
140.04
90.44
102.45
89.96
60.03
102.79
139.37
90.09
102.57
89.95
SSRL beam line
BL 9-2
BL 9-2
Peak
Peak
Inflection
Remote
BL 9-1
1.731
3.01
391,272
61,646
6.3
94.0 (89.0)
4.5 (2.1)
0.129 (0.272)
1.737
3.13
247,396
56,889
4.3
98.4 (97.2)
5.0 (2.1)
0.146 (0.325)
1.742
3.13
223,050
55,324
4.0
95.6 (93.9)
5.3 (2.2)
0.137 (0.299)
1.348
2.99
280,915
65,516
4.3
98.5 (97.8)
4.1 (2.2)
0.172 (0.307)
0.984
2.70
168,061
85,404
2.0
95.6 (95.6)
5.9 (1.9)
0.105 (0.337)
f′ (e)
f″ (e)
−5.51
4.37
−8.45
2.12
−0.29
2.57
8 site model at 4.5 Å
Phasing power
Figure of merit
1.65
2.05
0.63
0.66
32 site model at 3.5 Å
Phasing power
Figure of merit
1.26
1.49
0.53
0.38
FS4/21.6
FS4/33.5
FS4/22.9
FS4/33.7
FS4/20.2
FS4/32.6
FS4/20.2
FS4/33.1
—
APS/108.9
—
APS/109.8
—
APS/111.0
—
APS/106.8
Wavelength (Å)
Resolution (Å)
Observations
Reflections
Redundancy
Completenessa (%)
<I>/<σI>a
Rsymm(I)a
B. MAD phasing
C. Refinement
Resolution range (Å)
Reflections > 0.0 σF
R-factor
Rfree (% of data)
rmsd bond lengths (Å)
rmsd bond angles (deg.)
50.0 – 2.70
85,402
0.229
0.265 (4.7)
0.008
1.46b
D. Model
No. residues/<B-factor>c (Å2)
Subunit A
Subunit B
Subunit C
Subunit D
Subunit E
Subunit F
Subunit G
Subunit H
Water molecules
222/31.8
223/46.0
222/28.3
223/45.2
222/28.4
223/46.2
222/32.0
223/46.5
151/22.1
a
Values for highest resolution shell in parentheses.
Ramachandran plot: 87.9% of residues in most-favored regions; 11.6% in allowed regions; 0.5% in generously allowed regions; 0.1%
in disfavored regions.
c
The Wilson B value for 2.70 Å native data is 47.7 Å2.
b
sulfonated C-terminal residues, which become disordered in the absence of Trx. Hence, consideration of
the crystal structure together with biochemical data
provides a model for the conformational states of the
C-terminal residues during the overall reaction.
The above observations predict that when the
concentration of Trx is limiting, elevated concentra-
tions of APS should result in substrate inhibition. To
test the model, we assayed enzyme activity as a
function of APS and concentration of Trx. The
inhibitory effect of increasing the concentration of
APS above its Km is clear (Figure 6(a)), consistent
with APS acting as a competitive inhibitor, i.e. it
prevents recognition of the sulfonated C-terminal
159
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 5. Sites of trypsin proteolysis in M. tuberculosis and P. aeruginosa APS reductase. In the absence of substrate
(–APS, black arrows) both the Arg-loop and the C-terminal tail are cleaved; in the presence of equimolar substrate (+APS,
red arrows), formation of the thiosulfonate intermediate protects these structural elements, and only sites at the extreme N
and C termini are cleaved. (See Supplementary Data Figure 5 for SDS-PAGE analysis and peptide masses.)
residues by Trx. This interpretation is supported by
kinetic analysis with ten-fold additional Trx (Figure
6(b)). In this case, a higher concentration of Trx favors
sulfite release and regeneration of free enzyme. As a
result, additional APS is required to observe the
inhibitory effect (KiAPS increases from 7.4 μM to
81 μM). Thus, consistent with the crystallization
conditions, elevated concentrations of APS displace
the C-terminal thiosulfonated residues, which prevents productive association of Trx and inhibits the
enzyme. This model is summarized in Figure 6(c).
Iron-sulfur cluster
On the basis of the experimental phases, the
starting model for the cluster was a center of mass
only (Materials and Method); hence, the derived
cluster model is unbiased. The cluster is ligated by
four cysteine residues, Cys228 and Cys231, positioned at the tip of a β-loop, and the tandem pair,
Cys139 and Cys140, within a long, kinked helix, α6A
(Figure 7(a)). Despite constraints imposed by the
tandem coordination, the [4Fe-4S] cluster has tetrahedral symmetry and typical metric parameters. At
the same time, residues 136–143 exhibit normal αhelical geometry, although helix α6 is kinked where
Lys144 is oriented into the active site (Figure 4(a)). In
the context of a regular α-helix, the peptide bond
linking Cys139 and Cys140 straddles one face of the
cluster (Figure 7(b); and Supplementary Data Figure
6). Analysis of the cluster coordinates by the method
of circumcenters22 shows that the Sγ atoms are
displaced only slightly from ideal tetrahedral positions. The χ1,χ2 torsion angles about the Cα–Cβ and
Cβ–Sγ bonds are typical for Cys139, Cys228, and
Cys231, but the χ1,χ2 torsion angles for Cys140 are
(–g, cis), i.e. the Cα and Fe atoms are virtually
eclipsed. Consequently, accommodation of the
CysCys motif to the [4Fe-4S] cluster results in distortion of the Cys140 side-chain, and leads to steric
clashes between both cysteine residues and the cluster (Figure 7(b); and Supplementary Data Figure 6).
The [4Fe-4S](CysSγ)4 cluster sites, having a net
charge of –2, often exhibit NH…S hydrogen bonds
involving amide dipoles within ∼3.5 Å of S or Sγ.23
In P. aeruginosa APS reductase there is no such
interaction; rather, there are five charged and/or
polar NH…S or OH…S hydrogen bonds involving
side-chains, four representing strictly conserved
residues (Figure 7(a); and Supplementary Data
Figure 2). In particular, the CysCys motif interacts
with a pair of basic residues, Arg143 and Lys144, on
the next turn of helix α6; interactions also involve
Thr87 in the LDTG motif, and Trp246. A fifth
interaction might involve His136, which is His or
Arg in two-thirds of APS reductase sequences
(Figure 7(a); and Supplementary Data Figure 2).
Lastly, Cys231, and the face of the cluster opposite
the APS-binding site, pack against Phe131 and
Pro230, forming a hydrophobic interface. These
residues participate in a network of generally
conserved aromatic amino acids surrounding the
opposite side of the cluster, which includes Phe129,
Tyr132, Trp246, Trp247, and Trp248.
Substrate recognition
APS is bound to subunits B, D, F, and H in the two
tetramers in the asymmetric unit (Figure 4(c)). In the
absence of an ordered C-terminal segment, the active
site is accessible to solvent (Figure 8). APS fits into
the deep active site cavity with the phosphosulfate
moiety extending toward the protein surface, which
is slightly concave where it surrounds the active site
cleft. In addition to APS, the active site accommodates at least one water molecule adjacent to O3′ of
ribose. Conserved and semi-conserved residues line
the active site cavity. The adenosine moiety participates in four hydrogen bonds with main-chain
amide and carbonyl groups (Figure 9(a)). The 2′
hydroxyl group of the ribose sugar wedges between
strands β1 and β4, and the N-6 and N-1 atoms of
adenine interact with Leu85, the first residue in the
conserved LDTG motif on strand β2 (Figure 4(a)).
160
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 6. Dependence of APS reductase activity on the concentration of APS. (a) Reaction rate measured in the
presence of 1 μM thioredoxin. (b) Reaction rate measured in the presence of 10 μM thioredoxin. Data were fit with
equation (1b), derived from the inhibitory model in equation (1a) (see Materials and Methods). (c) Model for mobility of
the C-terminal tail during APS reduction. The C-terminal peptide of APS reductase (E) can be in an open or closed
conformation. Higher concentrations of Trx favor sulfite release and regeneration of free enzyme, resulting in a higher
apparent KAPS
. In the presence of excess APS, but absence of Trx, the enzyme is trapped in the inhibitory open form, as
i
observed in the crystals.
Complementary hydrogen bonding between adenine and the main-chain atoms of a β-strand residue
is a conserved feature of adenine nucleotide α
hydrolases.5,24 Hydrogen bonds within the conserved motifs cooperatively stabilize the β-strands
for recognition (Figure 9(a)). The adenine ring is
sandwiched between the side-chains of Leu85 and
Ser62; nearby residues not in van der Waals contact
are Phe83, Lys144, Val148 and Trp158.
A second aspect of the active site entails substrate
recognition by the P-loop (Figure 9(b)). The P.
aeruginosa APS reductase sequence Ser(62)-GlyAla-Glu-Asp(66) is strongly conserved in APS
reductases, and related to the P-loop motif in other
ATPases,5,24 and nucleotide-binding enzymes.25 In
particular, the E. coli PAPS reductase sequence,
SSSFGIQA, which contains the consensus motif
SXG,26 aligns with SFS-GAED in P. aeruginosa APS
reductase (Figure 3). It appears that Glu65 and
Asp66 in P. aeruginosa APS reductase, which interact
with three amide groups of the P-loop, and are
positioned above the dipole of the α3 helix, mimic
the interaction of the negatively charged 3′-phosphate group in PAPS reductase (Figure 9(b)).
Interaction between the APS phosphosulfate
moiety and APS reductase occurs via strictly
conserved basic residues, Lys144, Arg242, and
Arg245 (Figure 9(c)). Lys144 plays a role in cluster
interactions as well and, along with Arg143 (Figure
7(a)), these four basic residues balance the combined
negative charge of the phosphosulfate and [4Fe-4S]
(CysSγ)42– cluster. The phosphosulfate displays an
extended conformation with respect to C-5′ and O-5′
of the ribose, and is positioned opposite the [4Fe-4S]
cluster and Cys140. The sulfate group is somewhat
distal from the cluster and, while no atom intervenes, the shortest distances between sulfate oxygen
and Fe and Sγ of Cys140 are 7.03 Å and 6.09 Å,
respectively (Figures 7(a) and 9(c)). Two segments of
the Arg-loop, Arg(163)-Arg-Asp-Gln-Ser(167) and
Thr170-Arg171, are not in contact with APS,
although they represent consensus sequences for
APS and PAPS reductases, and Arg171 is one of only
six strictly conserved residues in both classes of
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
161
Figure 7. (a) The environment of the [4Fe-4S] cluster in P. aeruginosa APS reductase. The [4Fe-4S] cluster is ligated by
four cysteine residues at positions 139, 140, 228 and 231. Four conserved residues participate in charged or polar NH…S or
OH…S hydrogen bonds to inorganic S or cysteine Sγ atoms; Thr87, Arg143, Lys144 and Trp246. In addition, His136 may be
hydrogen bonded to Cys140. (b) Molecular details of the [4Fe-4S] cluster and its cysteine ligands in P. aeruginosa APS
reductase. The positions of H atoms, calculated on the basis of C and N atomic coordinates, indicate steric clashes (dotted
lines). Due to linkage of tandem cysteine residues in an α-helix, the χ2 torsion angle is cis (Supplementary Data Figure 6)
and the Cys140 Cα H atom is ∼2.6 Å from the inorganic S of the cluster. In addition, an H atom on Cβ of Cys139 is ∼2.3 Å
from an S atom. The sum of van der Waals radii for S and H is 3.00 Å.
enzymes (Supplementary Data Figure 2). Interactions with APS are summarized in Figure 9(d).
Discussion
By crystallography, we have captured P. aeruginosa
APS reductase in the state following the first step of
the catalytic cycle, in which the sulfite group of APS
is transferred to Cys256. In the absence of Trx, the
second step (reduction of thiosulfonate) does not
occur, but in the presence of excess substrate, APS
binds, and the thiosulfonated C-terminal peptide is
displaced from the active site. Consequently, the
analysis reveals a dynamic role for the C-terminal
tail in substrate binding and product release, such
that APS binding is accompanied by closure of the
C-terminal tail over the active site, bringing the
catalytic cysteine into proximity with the substrate
(Figure 6(c)). In the absence of a large excess of
substrate, the C terminus of the enzyme-
162
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 8. APS binds in a deep active site pocket of P. aeruginosa APS reductase such that only the sulfate group is
exposed. The solvent-accessible surface is depicted, and residues are colored by degree of sequence conservation
(Supplementary Data Figure 2) among 72 APS and PAPS reductases: green, most conserved; yellow, partial conservation;
white, variable). The orientation is similar to that in Figure 4(a).
thiosulfonate intermediate remains ordered, protecting it from proteolysis and preventing non-specific
hydrolysis to sulfate. However, the second half of
the reaction may require conformational rearrangement to make the enzyme-thiosulfonate intermediate accessible for reduction by Trx. Perhaps, the
reductase activity represented in the first half of the
reaction evolved from a protein ancestor that
interacted with APS or a related metabolite. Since
proteins that reduce disulfides are abundant in
ancient bacterial species, it may not have been
necessary for an ancestral reductase to reduce the
thiosulfonate bond in the second half of the
reaction.27 Instead, the glutathione-like GluCysGly
motif at the C terminus of APS reductase, which
includes the catalytic cysteine residue, could have
promoted recognition by glutaredoxin or Trx.
Mössbauer analyses of plant, P. aeruginosa and B.
subtilis enzymes established the presence of a [4Fe4S] cluster in APS reductase.2,16,28 However, the
unusual cysteine motif has spurred debate about the
identity of the fourth ligand coordinating the [4Fe4S] cluster.2,18,20 This work establishes that the [4Fe-
4S] cluster is ligated by four cysteine residues,
Cys228 and Cys231 on one side, and the tandem pair
Cys139 and Cys140 within an α-helix on the other
side (Figure 7(a)). Coordination by sequential
cysteine residues to a [4Fe-4S] cluster has been
observed in one other structure, the N2 cluster in the
Nqo6 subunit of Thermus thermophilus NADH:
ubiquinone oxidoreductase (complex I).29 These
tandem cysteine residues also reside within an αhelix. Further, complex I exhibits substrate-induced
conformational change in the Nqo6 subunit, suggesting that tandem cysteine coordination is associated with conformationally dynamic clusters.30
The [4Fe-4S] cluster and its four cysteine ligands
crosslink and stabilize the protein fold. Cysteine
mutagenesis and iron content analysis for M.
tuberculosis18 and P. aeruginosa20 APS reductases
demonstrate that the [4Fe-4S] cluster is required for
catalytic activity. In addition, high-resolution FTICR mass spectrometry data show that an intact
[4Fe-4S] cluster is required for interaction with AMP
or Trx, and that formation of the thiosulfonate
intermediate stabilizes the cluster toward oxidation
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 9 (legend on page 165)
163
164
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
and loss of activity. 18 Furthermore, resonance
Raman spectra of P. aeruginosa APS reductase
show a marked change in Fe-Sγ stretching modes
upon binding APS or AMP.20,31 Taken together,
these data imply an interaction between APS and
the cluster and, in this context it has been proposed
that the cofactor acts as a Lewis acid to facilitate
nucleophilic attack on the sulfate moiety.6,18 Yet, in
the current structure, the APS sulfate group is not in
direct contact with the cluster (Figure 9(c)). Given
the specific recognition of APS deep within the
active site (Figure 9(a) and (b)), interaction during
the catalytic cycle would require movement of the
cluster toward the sulfate group. This might entail
motion of helices α5 and α6A (i.e. residues ∼115–
143; Figure 4(a)) translating the tandem CysCys
ligands and the cluster. Conformational change
involving the cluster would not be surprising, as
the data indicate two other structural elements (Argloop and C-terminal tail) undergo substrate-associated conformational change. It would be consistent
also with differential labeling of the cysteine ligands
in cluster extrusion experiments, which are dependent upon the presence of ligand in the active site.18
In aconitase,32 4-hydroxybutyryl-CoA dehydratase,33 and radical SAM enzymes,34–38 substrates
bind to one Fe site of a [4Fe-4S]2+ cluster.23 In ferre-
doxin:thioredoxin reductase, one Fe of a [4Fe-4S]2+
cluster interacts with a fifth cysteine residue in a redox
active disulfide,39 and in heterodisulfide reductase
one Fe interacts with a substrate thiol.40,41 In order for
the APS reductase cluster to interact with APS, there
must be rearrangement relative to the observed
structure. One possibility, supported by biochemical,
spectroscopic, and mass spectrometry experiments
with M. tuberculosis APS reductase, is that a cysteine
residue in the tandem pair (Cys140 in P. aeruginosa
APS reductase) repositions itself while remaining
ligated to iron.6,18 Movement of Cys140 could be
facilitated by the steric clashes that arise from
CysCys ligation (Figure 7(b); and Supplementary
Data Figure 6). This model is supported by Mössbauer
data for plant and P. aeruginosa APS reductases, which
describe a diamagnetic [4Fe-4S]2+ cluster with Fe
subsites in a 3:1 ratio,2,16 and changes in Fe-Sγ
stretching modes upon addition of APS or AMP.20,31
PAPS reductases catalyze the same reaction as APS
reductases (Figure 1). E. coli PAPS reductase, crystallized in the absence of substrate, has a fold similar to
that of P. aeruginosa APS reductase with adenosine
recognition and P-loop motifs, an Arg-rich loop, and
a positively charged active site pocket.26 The E. coli
enzyme dimer corresponds to the AC pair in the P.
aeruginosa APS reductase tetramer (Figure 4(c)).
Figure 9 (legend on next page)
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
PAPS reductase also exhibits flexibility in the Cterminal tail as the last ordered residue observed is
His21626 (Figure 3). In PAPS reductase, the Arg-loop
is folded over the active site (Figure 10) when it is
unoccupied by substrate, rather than being displaced
onto the enzyme surface (Figure 8).
On the basis of the structural similarity between P.
aeruginosa APS and E. coli PAPS reductases, and the
fact that the residues involved in adenosine recognition are largely conserved among sulfonucleotide
reductases, it is likely that PAPS binds in a manner
similar to that of APS. Consequently, the additional
3′-phosphate group of PAPS may be accommodated
in the P-loop, where Glu65 is predominantly
replaced with Gln, and Asp66 is replaced with Ser
or Ala (Figures 3 and 9(b)). These substitutions
would allow interaction of the amide groups with a
3′ phosphate group and accommodate the bulkier
165
moiety. It appears that Glu65 and Asp66 define
substrate specificity in P. aeruginosa APS reductase,
compensating for the charge, volume, and hydrogen
bonding potential of a 3′ phosphate group, as if the
substrate were PAPS.
Sequence alignment highlights the evolutionary
divergence of APS versus PAPS reductases (Figure 3;
and Supplementary Data Figure 2). In APS reductases, 38 sequences indicate the presence of a [4Fe-4S]
cluster, whereas in 34 PAPS reductases, an alternative constellation of residues is present. Superposition of the P. aeruginosa APS and E. coli PAPS
reductase structures results in an rmsd of 6.67 Å for
188 pairs Cα atoms. Within this framework, only
three active site residues are invariant, Thr87/Thr79,
Lys144/Lys136, and Arg171/Arg164 (Figure 10).
The Arg appears to play a key role in alternate
conformations of the Arg-loop. The lysine interacts
Figure 9. (a) Recognition of the adenosine moiety of APS by conserved residues on strands β1, β2, and β4, which
participate in four main-chain hydrogen bonds with adenine and the ribose O2′ hydroxyl group. In addition, these residues
stabilize the conformation of the β-strands through hydrogen bonds within two conserved motifs (Leu85Asp86Thr87Gly88
and Thr160 Gly161). (b) The P-loop comprising conserved residues 60–66 connects strand β1 and helix α3 in the P. aeruginosa
APS reductase active site. Three amide groups hydrogen bond with Glu65 or Asp66, but in PAPS reductases these acidic
residues are replaced by Gln and Ala or Ser, respectively. The distance between Glu65 and the O3′ of ribose is 5.3 Å (cyan
dotted line). (c) Conserved basic residues in the vicinity of the active site interact with the phosphate and sulfate groups of
APS, or reside on the Arg-loop, comprising residues 162–175 between strands β4 and β5. The shortest distance between a
sulfate oxygen atom and Fe of the [4Fe-4S] cluster is ∼7.0 Å (cyan dotted line). (d) Summary of all active site contacts to APS
in subunit B of the asymmetric unit (PDB deposition 2GOY) plotted in two dimensions; hydrogen bond distances are
indicated in Å.
166
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Figure 10. Superposition of the structures of P. aeruginosa APS reductase and E. coli PAPS reductase showing the
positions of the [4Fe-4S] cluster and APS bound in APS reductase with respect to conserved active site residues in both
enzymes. Distinct conformations of the Arg loop are indicated, showing that the loop folds into the active site of PAPS
reductase in the absence of substrate. Arg164 (P. aeruginosa) and Arg171 (E. coli) are equivalent residues, conserved in 72
species of APS and PAPS reductases (Supplementary Data Figure 2).
with the substrate and appears capable of adopting
multiple conformations. The Thr interacts with a
cluster ligand (APS reductase), or with the conserved
residue Asp214 (PAPS reductase). In light of the
absence of a cofactor in PAPS reductase, perhaps the
interaction between Thr79 and Asp214 is stabilizing
in place of the cluster, like another involving Tyr131
with His216, which is conserved in 32 of 34 PAPS
reductases (Supplementary Data Figure 2). In contrast, different residues are conserved in association
with the [4Fe-4S] cluster, such as Arg143 and Trp246
(Figure 7(a)). An exception exists in the B. subtilis
enzyme, which utilizes both APS and PAPS as
substrates, and which contains an essential [4Fe-4S]
cluster.28 In this case, the sequence is APS reductaselike, except for the P-loop, which is more PAPS
reductase-like (Figure 3).
In addition to E. coli PAPS reductase,26 a search for
homologous folds in the Protein Data Bank42 yields
two significant alignments with P. aeruginosa APS
reductase: Pseudomonas syringae ATP sulfurylase,5
and E. coli GMP synthetase.24 Both enzymes are ATP
pyrophosphatases with adenine recognition and Ploop motifs. It is notable that ATP sulfurylase shares
additional similarities to APS reductase, including
an arginine-rich loop following the β4 strand, a
lysine residue oriented like Lys144/Lys136 in APS/
PAPS reductases, and mobility of C-terminal residues, which are positioned above the ATP site for
sulfate binding.5 Further, the involvement of a G
protein in ATP sulfurylase4,5 reflects a requirement
for conformational change during synthesis of APS,
reflecting, perhaps, the mobility of the C-terminal
tail, Arg-loop, and apparently the [Fe-S] cluster as
well, in APS reductase.
Materials and Methods
Materials used, and details of protein expression and
purification followed published procedures and are
described in the Supplementary Data.3,6,18
General kinetic analysis
APS reduction reactions were carried out as described.6,18 Reactions were performed at 30 °C and contained
5 nM APS reductase, 20 μM APS, 10 μM E. coli Trx, 5 mM
DTT in 50 mM Bis-Tris propane (pH 7.0) and 100 mM
NaCl. Reactions were followed to completion (≥5 halflives), except for very slow reactions. The initial linear
portion of the reaction (≤20% reaction) was used to
calculate the reaction rate. Kinetic data were measured in
at least two independent experiments and the standard
error was less than 15%.
Effect of small-molecule reductants on APS reductase
activity
The ability of various small-molecule reductants to
support APS reduction by M. tuberculosis APS reductase
was assayed as described above, except that 10 mM
reductant (DTT, GSH, reduced lipoic acid or dithionite)
was used in place of 10 μM E. coli Trx and 5 mM DTT
(Supplementary Data Table 1). Control reactions
contained 10 μM E. coli Trx, supplemented with 10 mM
DTT (required to regenerate reduced Trx).6
Sodium dithionite alone is not able to reduce the APS
reductase thiosulfonate intermediate (Supplementary Data
Table 1), but it is acid-labile, decomposing to sulfite and
other products, and sulfite is known to react with protein
disulfides to yield thiosulfonate. Under the conditions used
for anaerobic crystallization (see below), the relative concentrations of components at pH 6.5 were 40 μM protein,
167
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
2.5 mM APS, 2.3 mM DTT, and 2.5 mM sodium dithionite.
The data support the conclusion that the sulfate group
attached to Cys256 during crystallization is derived from
the substrate, APS, and not from sulfite via decomposition of
dithionite (Supplementary Data Figure 4a). In particular, the
reduced protein does not contain disulfides and the
anaerobic conditions would prevent their formation. In
addition, the enzyme reaction with APS is fast (Supplementary Data Figure 5), and a >60-fold excess of APS over
enzyme was present. On the other hand, if sodium
dithionite decomposition products were to react with free
sulfhydryl groups, reduced DTT in 115-fold excess over
Cys256 would quench this side-reaction.
Dependence of APS reductase activity on APS
concentration
The apparent Km APS for M. tuberculosis APS reductase
was determined at two concentrations of E. coli Trx, 1 μM
and 10 μM. The reaction rate was quantified as a function
of the concentration of APS. APS concentration-dependence reproducibly gave inhibition above 5 μM (1 μM Trx)
and 15 μM (10 μM Trx) APS. The concentration dependence was therefore fit to a model in which a second APS
that is inhibitory can bind to the E-Cys-Sγ–SO–3 complex
(equation (1b)), derived from the reaction scheme in
equation (1a). Using non-linear regression analysis, fits of
the data to this model gave a value of R2 > 0.98
(KaleidaGraph, Synergy Software, Reading, PA).
APS
v ¼ Vmax ½APS=Km
þ f½APSð1 þ ½APS=KiAPS Þg
ð1bÞ
Limited proteolysis of APS reductase
P. aeruginosa or M. tuberculosis APS reductase (50 μM)
was incubated with or without equimolar APS for 10 min
at room temperature (Supplementary Data Figure 5).
Subsequently, all samples were incubated on ice with
10 μg/ml of trypsin. At the time-points indicated, a 15 μl
sample of the proteolysis reaction was quenched by the
addition of SDS load dye and heated at 100 °C for 2 min.
Samples were analyzed by SDS-PAGE using 4%–12%
Criterion gradient gels (Bio-Rad, Hercules, CA). To map
trypsin digest sites, reactions identical with those described above were allowed to proceed for a total of 60 min
(P. aeruginosa APS reductase) or 90 min (M. tuberculosis APS
reductase) and stopped by freezing in liquid nitrogen.
Peptide fragments were separated by reversed-phase
chromatography on a Vydac 218TP54 protein and peptide
C18 column (The Separations Group, Hesperia, CA). The
molecular masses of peptide fragments were determined
by electrospray mass spectrometry and their identities
determined by analysis using GPMAW.43
Mass spectrometric analysis
All mass spectrometry data were acquired on a Bruker
(FT-ICR) mass spectrometer equipped with an actively
shielded 7 T superconducting magnet as described.6 Details, including the preparation and analysis of dissolved
crystal samples, are provided in the Supplementary Data.
Crystallization
APS reductase enzymes from four bacterial species were
expressed, purified, and screened for crystallization by
vapor diffusion. Samples of M. tuberculosis, M. smegmatis, R.
meliloti, and P. aeruginosa APS reductase were concentrated
under N2 and used for crystallization trials in an anaerobic
glove box (<1 ppm O2). Solutions were degassed, and
freshly prepared sodium dithionite was added to buffers to
a concentration of 5–10 mM. Protein solubility curves for
each APS reductase were determined using a matrix of
four protein concentrations versus precipitant concentration
(PEG3350 or ammonium sulfate) at four discrete pH values
(5.6, 6.5, 7.5, and 8.5). The curves indicated a sharp
transition in solubility for each protein at ∼2 mg/ml and
pH 7.5. These conditions were then used in sparse matrix
screening, which yielded the most hits with P. aeruginosa
APS reductase. Following refinement of the best conditions,
clusters of thin, blade-shaped, brown crystals could be
grown reproducibly within two weeks. Specifically, P.
aeruginosa APS reductase at 2.7 mg/ml in 50 mM Tris–HCl
(pH 8.0), 150 mM NaCl, 10% (v/v) glycerol, 5 mM DTT,
was mixed with of APS and sodium dithionite stock
solutions in a glove-box, and then mixed in 1:1 ratio with a
reservoir solution of 1.5 M ammonium sulfate, 100 mM
sodium cacodylate (pH 6.5). The resulting final concentrations in the crystallization drop (5 μl) were: 1.2 mg/ml of
APS reductase (40 μM), 22.5 mM Tris–HCl (pH 8.0),
67.5 mM NaCl, 4.5% glycerol, 2.3 mM DTT, 2.5 mM APS,
2.5 mM sodium dithionite, 0.75 M ammonium sulfate, and
50 mM sodium cacodylate (pH 6.5). To improve the size
and yield of single crystals, crystals were harvested and
used to seed 2 μl drops of the same solution, except at
0.65 M ammonium sulfate. Seeded drops were incubated
under paraffin oil at 24 °C in the glove box for one to two
weeks. Larger single crystals are uniformly birefringent.
For data collection, seeded drops under paraffin oil were
removed from the glove-box, and crystals were transferred
to 200 μl of cryoprotectant solution (0.8 M ammonium sulfate, 100 mM sodium cacodylate (pH 6.5), 5 mM sodium
dithionite, 25% glycerol). Within ∼10 min, the crystals
were transferred to nylon loops and flash-frozen in liquid N2.
During this time, no discoloration of the crystals was observed.
Crystallographic analysis
The structure was solved using Fe K-edge single
anomalous dispersion (SAD) and multiple anomalous
dispersion (MAD) data, combined with non-crystallographic symmetry averaging, solvent flattening, and
phase extension, and refined at 2.7 Å resolution (Table 1).
For initial diffraction experiments, duplicate data sets
were collected at BL 9-1. Data processing established that
the space group was P1, and not P21, with two APS
reductase tetramers in the unit cell (63% (v/v) solvent)
related by a pseudo-21 screw axis parallel with the b-axis.
For all crystals, Rmerge was >0.25 for data indexed and
scaled in monoclinic space groups, but <0.10 for space
group P1. All data sets indicated normal intensity statistics
without indication of twinning. A native set was collected
to 2.70 Å resolution (Table 1).
The expected anomalous and dispersive diffraction
ratios for four Fe atoms per 30 kDa of protein were
∼0.037 and ∼0.040, respectively. A SAD data set was
collected near the Fe K-edge; due to the triclinic symmetry,
frames collected over 720° achieved only ∼sixfold
redundancy (Table 1). The anomalous difference Patterson
map at 5.0 Å resolution contained 6–8σ peaks for the [Fe-S]
168
clusters; the coordinates for eight clusters were solved
using both CNS44 and ShelxD.45 The eight [Fe-S] clusters occur as two tetramers related by pseudo-21 symmetry; and cluster sites within each tetramer are arranged as a
trigonal antiprism with twofold symmetry only. This
defines fourfold NCS for the entire unit cell, and accounts
for the crystals being triclinic and not monoclinic.
A three wavelength MAD data set was collected (Table
1); due to decay, these data sets had lower redundancy
than the SAD data sets (540° total rotation/wavelength).
Using the eight cluster positions as eight pseudo-atom
sites at 4.5 Å resolution, MAD phases were calculated,
and the electron density map was subjected to fourfold
NCS averaging and solvent flattening using CNS.
Approximately 70% of the polypeptide chain in subunits
A and B was modeled into this map as poly(Ala) using
Xfit.46 The model was used for phase combination with
the 4.5 Å MAD phases, the resulting phases were
extended to 4.0 Å by NCS averaging and solvent flattening,
and more of the polypeptide was modeled. This process
was repeated at 3.7 Å and 3.5 Å. In the 3.5 Å map, cysteine
ligation to the [Fe-S] clusters in subunits A and B of the
NCS averaged map was readily apparent, together with
the density for a [4Fe-4S] cluster in each subunit. An
idealized [4Fe-4S] cluster was used to fit the density with
the directional restraints provided by the cysteine ligands,
resulting in a 32-site model for the individual Fe positions.
The individual Fe sites were refined and used to calculate
MAD phases to 3.5 Å resolution using CNS (Table 1). The
phase combination, phase extension, NCS averaging,
solvent flattening, and model building process was
repeated as before at 3.5 Å, 3.2 Å and 3.0 Å, until the
model for subunits A and B was essentially complete. The
model was then expanded to all eight subunits in the unit
cell, the NCS restraints were relaxed, and the refinement
proceeded normally to 2.70 Å using model based σAweighted 2|Fo| – |Fc| and composite omit maps in CNS.
A final, unbiased, σA-weighted |Fo| – |Fc| difference map
was used to model APS into subunits B, D, F, and H
(Supplementary Data Figure 3), and identify tightly bound
water molecules. Statistics for the refinement and final
model are summarized in Table 1.
Protein Data Bank accession code
Coordinates have been deposited with the RCSB Protein
Data Bank with accession code 2GOY.
Acknowledgements
This work was supported by National Institutes of
Health grants GM-48870 to CDS and AI-51622 to
CRB. KSC was supported by a postdoctoral fellowship from the Damon Runyon Cancer Research
Foundation (DRG-1783-03). We thank David S. King
of the HHMI Mass Spectrometry Laboratory at the
University of California, Berkeley for assistance with
mass spectrometry and peptide mapping. We thank
the generous assistance of staff personnel at the
Stanford Synchrotron Radiation Laboratory. SSRL is
a national user facility operated by Stanford University on behalf of the U.S. Department of Energy,
Office of Basic Energy Sciences. The SSRL Structural
Molecular Biology Program is supported by the
P. aeruginosa Adenosine 5′-Phosphosulfate Reductase
Department of Energy, Office of Biological and Environmental Research, and by the National Institutes
of Health, National Center for Research Resources,
Biomedical Technology Program, and the National
Institute of General Medical Sciences.
Supplementary Data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jmb.2006.08.080
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Edited by M. Guss
(Received 9 June 2006; received in revised form 19 August 2006; accepted 28 August 2006)
Available online 1 September 2006
Supplementary Data
Substrate Recognition, Protein Dynamics, and Novel Iron-Sulfur Cluster in
Pseudomonas aeruginosa Adenosine Phosphosulfate Reductase
Justin Chartron, Kate S. Carroll, Carrie Shiau, Hong Gao,
Julie A. Leary, Carolyn R. Bertozzi, C. David Stout
1
Materials and Methods
Materials
Nonradioactive APS was purchased from Biolog Life Sciences Institute, ≥ 95%
(Bremen, Germany). [35S]SO42- (specific activity 1491 Ci/mmol) was obtained from MP
Biochemicals (Irvine, California, United States). Molecular biology grade DTT was from
Invitrogen (Carlsbad, California, United States). E. coli Trx protein was purchased from
EMD Biosciences (San Diego, California, United States). DPCC-treated trypsin was
purchased from Sigma. Depending upon availability, PEI-Cellulose TLC plates (20 cm x
20 cm) were purchased from J.T. Baker (Phillipsburg, New Jersey, United States) or
EMD Biosciences.
35
S-labeled APS and PAPS were prepared by incubating
[35S]Na2SO4, ATP, ATP sulfurylase (Sigma), inorganic pyrophosphatase (Sigma) and
recombinant APS kinase together as previously described1. All other chemicals were
purchased from J. T. Baker and were of the highest purity available (≥ 95%).
Protein Expression and Purification
The gene encoding the M. tuberculosis APS reductase was amplified from H37Rv M.
tuberculosis genomic DNA and cloned in a protein expression vector as previously
described2.
The gene encoding Mycobaterium smegmatis APS reductase was
amplified from M. Smegmatis genomic DNA and the gene encoding P. aeruginosa APS
reductase was amplified from P. aeruginosa genomic DNA ATCC 47085D (ATCC,
Manassas, Virginia, United States) as previously described1. Briefly, APS reductase
genes were amplified via PCR and cloned into the pET24b vector (Novagen) using the
5’ Nde I and 3’ Xho I restriction enzyme sites.
2
The expression plasmid encoding
Rhizobium meliltoi APS reductase was generated as previously described3.
Proteins were expressed by transforming a reductase-containing plasmid into
BL21(DE3) cells (Novagen) grown on LB-agarose containing 50 µg/ml kanamycin. An
isolated colony was grown in 5 ml of LB broth containing 50 µg/ml kanamycin. The
culture was grown at 37 °C overnight. This culture was used to inoculate 1 L of LB
broth containing 50 µg/ml kanamycin. The culture was grown with shaking (250 rpm) at
37 °C to an OD of 0.6, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a
final concentration of 0.4 mM and the cells harvested after 4 h. Subsequently, 1 L of
cells were collected by centrifugation and resuspended in 30 ml of lysis buffer (20 mM
sodium phosphate, pH 7.4, 0.5 M sodium chloride (NaCl), 10 mM imidazole, 1mM
methionine) together with an EDTA-free protease inhibitor tablet (Roche, Indianapolis,
Indiana, United States). After sonication, DNase and RNase (Sigma) were added to the
lysate at 10 µg/ml and 5 µg/ml, respectively, and stirred for 10 min on ice. The cell
lysate was cleared by centrifugation and the supernatant was applied to a 5-ml HiTrap
Chelating column (Amersham, Piscataway, New Jersey, United States). The column
was washed with ten column volumes in 20 mM phosphate, pH 7.4, 0.5 M NaCl and 50
mM imidazole, and was eluted with 20 mM phosphate, pH 7.4, 0.5 M NaCl and 250 mM
imidazole. Fractions containing the desired protein were pooled and concentrated using
Amicon 10,000-Da molecular weight cut-off centrifugal filters (Millipore, Billerica,
Massachusetts, United States) before injection onto a 16/60 Superdex 200 prep grade
gel filtration column. The standard gel filtration buffer was 50 mM Tris-HCl, pH 8.0, 10%
Glycerol, 5 mM DTT with ionic strength adjusted to 150 mM with NaCl.
3
Fractions
containing APS reductase were pooled, aliquoted into single use portions, snap-frozen
in liquid nitrogen and stored at -80 °C.
Protein concentrations were determined
precisely by quantitative amino acid analysis (AAA Service Laboratory, Boring, Oregon,
United States).
Mass Spectrometric Analysis of Dissolved Crystals and Enzyme-Substrate Complexes
Solutions were infused at a rate of 2 µl/min into an Apollo electrospray source (Bruker,
Billerica, MA), operated in the positive mode. The syringe and spray chamber were
wrapped with ice bags to maintain low temperature, in order to prevent the protein from
precipitating. All ions were collected using gated trapping and detected using chirp
excitation. Broad band data were acquired using an average of 16-64 time domain
transients containing 32 K or 1 M data points. The original time domain free induction
decay (FID) spectra were zero filled, Gaussian-multiplied and Fourier transformed. All
data were acquired and processed using Bruker Xmass version 6.0.0 software. The
parameters of the electrospray ionization (ESI) source, ion optics, and cell were tuned
for the best signal-to-noise ratio and were maintained for systematic experiments.
Crystals from 10 drops were harvested and centrifuged, yielding a brown pellet, which
was washed three times in reservoir solution. The pellet dissolved readily in 10 µl of 2
mM β-octyl glucoside in H2O, and the resulting solution was discernibly light greenbrown in color.
The solution was incubated overnight with Biobeads to remove
detergent and frozen.
Subsequently, this sample was dialyzed against 50 mM
ammonium acetate using Amicon 10,000-Da molecular weight cut-off centrifugal filters
4
to remove residual salt and detergent, and the protein concentration was determined
(20 µM). This solution was then diluted with 80:20 acetonitrile:water containing 1%
formic acid for mass spectrometry analysis. The derived molecular weights correspond
to the full length polypeptide plus SO3– (31359.8 Da; theoretical 31360.1 Da) and the
same minus the three N-terminal amino acids (31018.2 Da; theoretical 31018.6 Da)
(50% of the protein used for crystallization lacked these residues) (Supplementary
Figure 4(a)). The data indicate that the enzyme in the crystals is quantitatively sulfated.
For enzyme-substrate incubation experiments with M. tuberculosis APS reductase
(Supplementary Figures 4(b), (c)), appropriate volumes of enzyme (after buffer
exchange to ammonium acetate) and APS were mixed in ammonium acetate buffer and
the mixtures were chilled on ice for at least 15 min before being introduced into the
mass spectrometer.
Supplementary References
1.
Carroll, K. S., Gao, H., Chen, H., Stout, C. D., Leary, J. A. & Bertozzi, C. R.
(2005). A conserved mechanism for sulfonucleotide reduction. PLoS Biology 3,
e250.
2.
Williams, S. J., Senaratne, R. H., Mougous, J. D., Riley, L. W. & Bertozzi, C. R.
5
(2002). 5'-Adenosinephosphosulfate lies at a metabolic branch point in
mycobacteria. J. Biol. Chem. 277, 32606-32615.
3.
Schwedock, J. & Long, S. R. (1990). ATP sulphurylase activity of the nodP and
nodQ gene products of Rhizobium meliloti. Nature 348, 644-647.
4.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
(1997). The CLUSTAL_X windows interface: flexible strategies for multiple
sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 48764882.
Supplementary Figure Legends
Supplementary Figure 1.
Routes of sulfate assimilation. Inorganic sulfate is
adenylated by ATP sulfurylase (a) to form APS. Higher plants and the majority of sulfate
reducing bacteria use APS as their source of sulfite (c1→d→e). In some organisms,
APS kinase (b) phosphorylates APS at the 3’-hydroxyl to form PAPS for use as a sulfate
donor for sulfotransferases or as a source of sulfite. The lower pathway of sulfate
reduction (c2→d→e) is utilized by γ-proteobacteria such as E. coli and some fungi.
Depending on the organism, APS or PAPS is reduced to sulfite by APS reductase (c1)
and PAPS reductase (c2), respectively. Sulfite is reduced to sulfide by sulfite reductase
(d) and incorporated into cysteine by O-acetylserine-(thiol) lyase (e). Important
6
metabolites such as methionine and coenzyme A are, in turn, synthesized from
cysteine.
Supplementary Figure 2. Structure based sequence alignment of 38 APS reductases
from prokaryotes and plants, and 34 PAPS reductases from prokaryotes and
eukaryotes. The Clustal X (v1.81) Multiple Sequence Alignment program4 was used
first to define profiles for each group, then to align all APS reductase sequences
(species names in square brackets) and all PAPS reductase sequences separately, and
then to align all 72 sequences as one group. The figure is color coded by residue
property.
The bar graph indicates the degree of conservation per position and is
included in Figure 3 under an abbreviated sequence alignment for four species.
Supplementary Figure 3. Electron density for APS in each of four subunits of P.
aeruginosa APS reductase. The unbiased σA-weighted |Fo|-|Fc| map, based on the
final model at 2.70 Å resolution, is contoured at 2.5 σ and 5.0 σ.
Supplementary Figure 4. (a) ESI FT-ICR mass spectrum of dissolved crystals of P.
aeruginosa APS reductase demonstrating quantitative sulfonation of enzyme in the
crystals. The two molecular weights correspond to the full-length enzyme plus SO3 ,
−
and the same minus the three N-terminal amino acids (~50% of the protein used for
crystallization lacked these N-terminal residues). These molecular weights represent
the apo-enzymes with the noncovalently bound [4Fe-4S] cluster dissociated from the
protein during preparation of the sample for mass analysis (see Methods). (b) ESI FT-
7
ICR mass spectrum of 10 µM M. tuberculosis APS reductase with 20 µM APS in
ammonium acetate showing that in the presence of excess substrate that the product
AMP can be displaced, and that the sulfonated enzyme (E-SO3–) can also bind APS, as
observed in the crystals. Previously, 15 µM enzyme was incubated with 10 µM APS;
under those conditions, only the sulfonated enzyme with AMP bound was observed
(Figure 7(a) of ref. 7). (c) ESI mass spectrum of the same mixture dissolved in 80:20
acetonitrile:water containing 1% formic acid, illustrating the release of the noncovalently
bound nucleotide and the iron-sulfur cluster. The inset shows the deconvoluted mass of
the thiosulfonate intermediate in the apo-form.
Supplementary Figure 5.
Partial trypsin proteolysis of M. tuberculosis (a) and P.
aeruginosa (b) APS reductase showing protection of the C-terminal tail and the Arg-loop
upon formation of the thiosulfonate intermediate at equimolar concentration. The time
course of the trypsin digestion is shown in the presence (+APS) and absence (-APS) for
each enzyme. In (a) M. tuberculosis APS reductase (50 µM active site concentration)
was incubated with or without 50 µM APS for 10 min at RT, and trypsin was added at a
final concentration of 10 µg/ml and incubated at 4 °C.
In (b) P. aeruginosa APS
reductase (40 µM active site concentration) was incubated with or without 40 µM APS
for 10 min at RT, and trypsin was added at a final concentration of 10 µg/ml and
incubated at 4 °C. All samples were analyzed by SDS-PAGE using a 4-12% gradient
Criterion gel.
Trypsin digest fragments were purified by reverse phase HPLC and
analyzed by electrospray mass spectrometry.
In the presence of APS, the starred
fragments, HR/G – End* for M. tuberculosis and ER/N – SK/A* for P. aeruginosa,
8
represent the mass of the peptide fragment plus an additional 80 Da for the covalent
sulfite adduct. Full length M. tuberculosis APS reductase without N-terminal Met, is
28,356.87 Da; full length P. aeruginosa APS reductase is 31,279.6 Da.
Supplementary Figure 6. Electron density for the [4Fe-4S] cluster and its Cys ligands
in Subunit B of P. aeruginosa APS reductase. The σA-weighted 2|Fo|-|Fc| map, based
on the final model at 2.70 Å resolution, is contoured at 1.0 σ and 5.0 σ. The Cα-Cβ-SγFe torsion angle for Cys140 is indicated; this angle is cis (+10°) so that the Cα and Fe
atoms are eclipsed and only 3.5 Å apart.
9
Supplementary Table 1
Effect of reductants on APS reductase activity a
Reductant
b
E°ʹ′, mV
Activity (pmol/min)
Thioredoxin
-260
40
GSH
-230
≤ 0.1
Reduced lipoic acid
-290
≤ 0.1
DTT
-330
≤ 0.1
Dithionite
-527
≤ 0.1
c
(a) Rate of APS reduction measured with various reductants. Each value reflects the
average of at least two independent experiments, and the standard deviation was less
than 15% of the value of the mean.
(b) 10 µM thioredoxin or 10 mM chemical reductant was used in each assay (see
Methods).
(c) Due to the slow nature of the reactions measured with chemical reductants,
reported rates are considered upper limits.
10